Unique Physical Properties and Interactions of the Domains of Methylated DNA Binding Protein 2

Abstract

MeCP2 is a methyl CpG binding protein whose key role is the recognition of epigenetic information encoded in DNA methylation patterns. Mutation or mis-regulation of MeCP2 function leads to Rett syndrome as well as a variety of other Autism Spectrum Disorders. Here, we have analyzed in detail the properties of six individually expressed human MeCP2 domains spanning the entire protein with emphasis on their interactions with each other, with DNA, and with nucleosomal arrays. Each domain contributes uniquely to the structure and function of the full-length protein. MeCP2 is ~60% unstructured, with nine interspersed α-Molecular Recognition Features (α-MoRFs), which are polypeptide segments predicted to acquire secondary structure upon forming complexes with binding partners. Large increases in secondary structure content are induced in some of the isolated MeCP2 domains and in the full-length protein by binding to DNA. Interactions between some MeCP2 domains in cis and trans seen in our assays, likely contribute to the structure and function of the intact protein. We also show that MeCP2 has two functional halves. The N-terminal portion contains the methylated DNA binding domain (MBD) and two highly disordered flanking domains which modulate MBD-mediated DNA binding. One of these flanking domains is also capable of autonomous DNA binding. In contrast, the C-terminal portion of the protein which harbors at least two independent DNA binding domains and a chromatin specific binding domain is largely responsible for mediating nucleosomal array compaction and oligomerization. These findings lead to new mechanistic and biochemical insights regarding the conformational modulations of this intrinsically disordered protein, and its context-dependent in vivo roles.

DNA Binding Restricts the Intrinsic Conformational Flexibility of Methyl CpG Binding Protein 2 (MeCP2)

“…Accordingly, we envision that the full-length MeCP2 polypeptide chain rapidly samples many different secondary structures and equilibrates between multiple tertiary structures, even when bound to DNA. In this regard, various domain-domain interactions in cis and trans have been detected previously (35,47), consistent with the conformational malleability of full-length MeCP2 observed by H/DX.…”

“…Previous studies have demonstrated that the isolated intervening domain, transcriptional regression domain, and C-terminal domain ␣ of MeCP2 are each capable of binding unmethylated DNA in native gel mobility shift assays (47). However, as discussed above, the H/DX experiments find no evidence of protection outside the MBD when MeCP2 is bound to unmethylated DNA.…”

“…The NMR solution structures (36) and the crystal structure (37) provide clear views of a favored folded state of the molecule, but to understand the dynamics of unfolding/refolding of the MBD, a measurement that is capable of characterizing dynamic protein behavior, such as the H/DX approach described here, needed to be employed. In this regard, CD analysis has shown that MeCP2 and many of its isolated domains gain secondary structure content when bound to unmethylated and methylated DNA (35,47). These averaged measurements are likely assaying the same global and local decreases in MBD flexibility observed by H/DX.…”

“…deconvolution of CD data yields 60 -65% unstructured content, and the protein sediments as if it were a random coil (34). Prediction algorithms (PONDR-VXT, FoldIndex) also yield estimates of approximately 65% disorder, with short regions of predicted order interspersed throughout an otherwise intrinsically disordered polypeptide chain (34,47). MeCP2 is composed of six biochemically defined domains: the N-terminal domain, the MBD, the intervening domain, the transcriptional repression domain, and the C-terminal domain ␣ and ␤ (supplemental Fig.…”

Coil‐to‐helix transitions in intrinsically disordered methyl CpG binding protein 2 and its isolated domains

“…IDPs often gain secondary structure concomitant with macromolecular interactions. 2,[16][17][18] In the case of MeCP2, the NTD interacts with the protein HP1, 19 the TRD with many different co-repressor proteins [22][23][24][25][26][27] and unmethylated DNA, 8,41 and the CTD with unmethylated DNA, 41 chromatin, 41 and RNA splicing proteins. 20,21 Moreover, some proteins interact with multiple MeCP2 domains, e.g., PU.1 binds to the NTD and TRD, 28 CDKL5 binds to the TRD and CTD.…”